In dogs, it is thought that the ability to absorb essential amino acids such as tryptophan and leucine may be limiting to cellular metabolism. Recent research designed to characterize the amino acid absorption capacity of the brush border (lumen facing) membranes of dog enterocytes suggests that peptide absorption may be particularly important given the relatively low amount of free amino acid transport capacity that was observed. Buddington R K, Paulsen D B. Development of the Canine and Feline Gastrointestinal Tract. In: Reinhart G A, Carey D P, eds. Recent Advances in Canine and Feline Nutrition, Vol. II: 1998 Iams Nutrition Symposium Proceedings. Wilmington: Orange Frazer Press, 1998; 195-215. Data collected from studies designed to understand the quantitative importance of free versus peptide amino acids in other monogastric animals strongly indicates that peptide-bound amino acids account for the majority of amino acids absorbed by enterocytes from the intestinal lumen (Matthews, D M. Protein Absorption, Development and Present State of the Subject, New York: Wiley-Liss, 1991.) and that the rate of peptide-derived amino acid absorption is faster than that by equivalent amounts of free amino acids. Ohkohchi N, Andoh T, Ohi R, Mori S. Defined formula diets alter characteristics of the intestinal transport of amino acid and peptide in growing rats. J Pediatr Gastroenterol Nutr 1990 May; 10(4):490-6.
Two types of peptide transporters have been cloned from monogastric animals. Liang R, Fei Y J, Prasad P D, Ramamoorthy S, Han H, Yang-Feng T L, Hediger M A, Ganapathy V, Leibach F H. Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J Biol Chem 1995 Mar. 24; 270(12):6456-63. Liu W, Liang R, Ramamoorthy S, Fei Y J, Ganapathy M E, Hediger M A, Ganapathy V, Leibach F H. Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney. Biochim Biophys Acta 1995 May 4; 1235(2):461-6. PepT1 is an H+-dependent, low-affinity (mM), high-velocity, transporter that is predominately localized primarily to the brush border membranes of mature enterocytes of intestinal villi. PepT2 is an H+-dependent, high-affinity (xcexcM), low-velocity, transporter that is expressed in the greatest abundance in the apical membranes of renal proximal tubular epithelial cells. An important feature of the peptide transporters is their ability to recognize and transport most di- and tripeptides, albeit with a range of relative affinities for different peptides. In addition, both transporters recognize the xcex2-lactam antibiotics, and carboxyl-terminal modified free amino acids. The physiologic functions of these transporters are thought to be to absorb di- and tripeptides from the digesta and from the blood, respectively. Although molecular evidence has not been acquired, there is strong biochemical evidence for a different peptide transport protein that functions in the basolateral membrane of these cells. Saito H, Inui K I. Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2. Am J Physiol 1993 August; 265(2 Pt 1):G289-94. Thwaites D T, Brown C D, Hirst B H, Simmons N L. Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by the expression of H+-coupled carriers at both the apical and basal membranes. J Biol Chem 1993 April 15; 268(11):7640-2.
Research with Caco-2 cells indicates that PepT1 transporter mRNA, protein, and activity increases in a manner consistent with a direct effect of increased extracellular substrate concentrations. Walker D, Thwaites D T, Simmons N L, Gilbert H J, Hirst B H. Substrate upregulation of the human small intestinal peptide transporter, hPepT1. J Physiol 1998 Mar. 15; 507(Pt 3):697-706. In contrast to mRNAs for essential amino acid transporters, intestinal studies show that the expression of peptide transporter mRNA increases in response to increased dietary protein. Erickson R H, Gum J R Jr, Lindstrom M M, McKean D, Kim Y S. Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs. Biochem Biophys Res Commun 1995 Nov. 2; 216(1):249-57. Similarly, expression in intestinal mucosa of PepT1 mRNA and protein increases in response to tissue trauma, whereas the MRNA for essential amino acid transporters decreases. Tanaka H, Miyamoto K I, Morita K, Haga H, Segawa H, Shiraga T, Fujioka A, Kuoda T, Taketani Y, Hisano S, Fukui Y, Kitagawa K, Takeda E. Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury. Gastroenterology 1998 April; 114(4):714-23.
Few studies have been conducted to evaluate the potential for the dog to absorb quantitatively significant amounts of essential amino acids in the form of small peptides, and whether this capacity can be regulated by substrate supply. Accordingly, there is still a need to evaluate the potential for the absorption of peptide-bound leucine and tryptophan by putative canine peptide transporters. It would thus be desirable to provide the nucleic acid sequence encoding canine PepT1. It would also be desirable to provide mRNA transcripts corresponding to cPepT1. It would further be desirable to characterize the function of cPepT1 by GlySar uptake and identify di- and tripeptides well recognized by cPepT1, as well as characterize the effect of supplemental peptide substrate on the transport capacity of canine PepT1 (cPepT1).
The present invention provides novel isolated and purified nucleic acids (RNA or DNA) encoding, or complementary to, canine PepT1 (cPepT1). The nucleic acid may be SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:20 or may be a nucleic acid that hybridizes under moderate or stringent hybridization conditions to any of these sequences. Also provided are peptides encoded by these nucleic acids, such as SEQ ID NO: 13 or SEQ ID NO:21.
The present invention also provides a method for determining canine PepT1-transportability of a peptide, or method for determining a peptide with beneficial nutritional property in an animal, comprising providing an immortalized kidney distal tubule epithelial (Madin-Darby Canine Kidney (MDCK)) cell and a peptide having 2 to 10 amino acids, and determining the amount of the peptide transported into the cell, wherein the amount correlates with the canine PepT1-transportability of the peptide. A peptide with beneficial nutritional properties in an animal is a peptide that contains at least one essential amino acid that is absorbed at a rate higher than the rate of the amino acid if it were free rather than in a peptide-bound form. The peptide may be a dipeptide, tripeptide, or tetrapeptide such as, for example, GlySar, GlyGly, AlaHis, xcex2-AlaHis (carnosine), GlnGln, GlyMet, LeuMet, LeuTrp, MetLeu, MetMet, MetPhe, MetPro, TrpLeu, TrpTrp, GlnGlu, MetGlu, MetLys, TrpGly, MetGlyMetMet (SEQ ID NO:10), TrpGlyGly, LeuArg, ArgLeu, GlyLeu, or ArgTrp. The cell used in the method may be in medium at a pH of between about 5 and 8; or at a pH of about 5.5 to 7.5, or even at about 6 to 6.5. The peptide may be present at a concentration of about 10 nm to about 50 mM.
The characterization of GlySar uptake by immortalized MDCK cells demonstrates that MDCK cells express PepT1-like activity, confirming detection of PepT1 MRNA expression by MDCK cells and the use of MDCK cells as a model to characterize the biochemical function of canine PepT1.
The cPepT1 of the present invention is also capable of recognizing a variety of di- and tripeptides, including those that contain the essential amino acids leucine and tryptophan, considered to be of especial importance to canine nutrition. In addition, H+-dependent peptide transport in cultured MDCK cells can be stimulated by at least two of PepT1 substrates, GlySar and carnosine. Moreover, H+-dependent uptake of GlySar by MDCK is sensitive to nutrient deprivation and Insulin-like Growth factor I (IGF-I).
The present invention further provides a dietary composition with improved nutritional benefit for an animal comprising at least one peptide identified by the method described above.
The present invention provides a process for altering the absorption of essential amino acids in an animal, such as a dog, comprising the steps of feeding the animal a diet containing the dietary composition described above; and maintaining the animal on the diet for a sufficient period of time to allow the composition to be absorbed by the digestive system of the animal. The diet may comprise about 20 to about 30% crude protein, about 10 to about 20% fat, and about 3 to about 10% dietary fiber.
As used herein, the term xe2x80x9ccPepT1xe2x80x9d includes variants or biologically active or inactive fragments of this transport protein. A xe2x80x9cvariantxe2x80x9d of the polypeptide is a cPepT1 protein that is not completely identical to a native cPepT1 protein. A variant cPepT1 protein can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acid. The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A xe2x80x9cconserved substitutionxe2x80x9d is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 common amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. Stryer, L. Biochemistry (2d edition) W. H. Freeman and Co. San Francisco (1981), p. 14-15; Lehninger, A. Biochemistry (2d ed., 1975), p. 73-75. It is known to those of skill in the art that transport of other, less common, amino acids such as hydroxylysine, or derivatives of any one of the 20 common amino acids listed above would also be within the scope of this invention.
It is known that variant polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that result in increased bioactivity. Alternatively, amino acid substitutions in certain polypeptides may be used to provide residues that may then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.
One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity. It is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to conduct substitutions of amino acids where these values are xc2x12, with xc2x11 being particularly preferred, and those with in xc2x10.5 being the most preferred substitutions.
The variant cPepT1 protein comprises at least seven amino acid residues, preferably about 20 to about 700 residues, and more preferably about 50 to about 700 residues, wherein the variant cPepT1 protein has at least 50%, preferably at least about 80%, and more preferably at least about 90% but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native cPepT1 protein.
The amino acid sequence of the variant cPepT1 protein corresponds essentially to the native cPepT1 protein amino acid sequence. As used herein xe2x80x9ccorrespond essentially toxe2x80x9d refers to a polypeptide sequence that will elicit an absorption value substantially the same as the absorption stimulated by native cPepT1 protein. Such absorption may be at least 60% of the level generated by native cPepT1 protein, and may even be at least 80% of the level generated by native cPepT1 protein.
A variant of the invention may include amino acid residues not present in the corresponding native cPepT1 protein, or may include deletions relative to the corresponding native cPepT1 protein. A variant may also be a truncated xe2x80x9cfragmentxe2x80x9d as compared to the corresponding native cPepT1 protein, i.e., only a portion of a full-length protein. cPepT1 protein variants also include peptides having at least one D-amino acid.
The cPepT1 protein of the present invention may be expressed from an isolated nucleic acid (DNA or RNA) sequence encoding the cPepT1 protein. Amino acid changes from the native to the variant cPepT1 protein may be achieved by changing the codons of the corresponding nucleic acid sequence. xe2x80x9cRecombinantxe2x80x9d is defined as a peptide or nucleic acid produced by the processes of genetic engineering. It should be noted that it is well-known in the art that, due to the redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon, and still result in an identical amino acid sequence. The terms xe2x80x9cprotein,xe2x80x9d xe2x80x9cpeptidexe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used interchangeably herein.